AMPK Methods and Protocols

23. Plate bone marrow cells in 100 mm tissue culture plates
    (10 mL per plate) (seeNote 8).


24. Incubate the cells in a humidified 5% CO 2 incubator at 37C
    for 7–8 days (seeNote 9).


25. Following 7–8-day incubation, remove the media, wash once
    with 1PBS, and then gently scrape cells in 2–3 mL BMDM
    medium.


26. Dilute cell suspension 1:1 in trypan blue and count the number
    of live cells using a hemocytometer.


27. Plate cells as necessary for future experiments.

3.2^3 H-Acetate
Incorporation into
Cholesterol
(Cholesterol
Synthesis)

1. After BMDM have differentiated, seed cells at 5 105 in
   12-well plates.


2. The following day, remove medium and wash the cells with 1
   PBS (seeNote 10).


3. Add 1 mL/well of BMDM medium containing 0.5 mM
   sodium acetate and 1μCi/mL^3 H-sodium acetate.


4. Add 2μL of a 50 mM A-769662 stock for a final concentration
   of 100μM. Vehicle-treated wells should receive 2μL of DMSO
   (other AMPK activators can be substituted as required;see
   Table1 for partial list of AMPK agonist drugs previously used
   in macrophages) (seeNote 11).


5. Incubate cells in a humidified 5% CO 2 incubator at 37C for
   the desired time (usually 4–16 h) (seeNote 12).


6. Remove radioactive medium and wash twice with 1PBS.


7. Scrape cells in 250μL of PBS and remove 200μL to a new
   1.5 mL centrifuge tube (keep the remaining volume for protein
   determination).


8. Add 750μL of methanol/chloroform (2:1) and vortex vigor-
   ously for 10 s.


9. Let sit on ice for ~10 min.


10. Add 250μL of chloroform and vortex.


11. Add 250μL of ddH 2 O, vortex briefly, and spin at 3000gfor
    5 min.


12. Remove lower lipid phase and evaporate to dryness under a
    stream of nitrogen gas (seeNote 13).


13. Resuspend samples in 20μL of chloroform and spot onto a
    TLC plate with a Hamilton syringe, including a nonradioactive
    cholesterol standard (preferably in each lane).


14. Develop the TLC plate in a previously equilibrated TLC tank
    with heptane/isopropyl ether/acetic acid (60:40:3), until the
    solvent front is approximately 2 cm from the top of the plate.

Assessing Macrophage Cholesterol Homeostasis 485